The emergence of reduced susceptibility to ciprofloxacin among Salmonella enterica serotype Typhi and serotype Paratyphi A leading to clinical failure of treatment poses a great therapeutic challenge. The mechanism of fluoroquinolone resistance in clinical isolates of S. Typhi and S. Paratyphi A is not very well documented. The present study was carried out with the objective of molecular characterization of reduced quinolone susceptibility amongst the strains of S. Typhi and S. Paratyphi A isolated from the patients with enteric fever during January, 2000, to April, 2003, in a North Indian hospital. A total of 422 culture-positive cases of enteric fever were reported to the hospital during the period of study, of which S. Typhi was isolated from 350 cases and S. Paratyphi A from 72 cases. The antimicrobial susceptibility of these strains was determined by disk diffusion and agar dilution method according to NCCLS guidelines, and E-test method. A total of 140 randomly selected strains, isolated during the years 1993-1999, that were available from the laboratory stocks were also studied to compare with the present strains. To study the quinolone susceptibility, the strains were divided into nalidixic acid sensitive (NAS), nalidixic acid intermediate resistant, (NAI) and nalidixic acid resistant (NAR) on the basis of susceptibility to nalidixic acid. Clinical history was available from 174 patients, of which 93 needed hospitalization due to severe disease. Of these, 82 patients were infected with NAR strains and 22 patients had a documented evidence of clinical failure to ciprofloxacin therapy. The patients infected with NAR strains were younger and had a significantly longer duration of fever (p value < 0.05) than those infected with NAS strains. It was observed that the proportion of NAR strains increased gradually over the years. These strains had a significantly higher range of MIC of ciprofloxacin (0.023-1.0 microg/ml) as compared to the NAS strains (0.002-0.125 microg/ml) (p value < 0.05). The sequencing of quinolone resistance determining region (QRDR) of the gyrA gene showed the presence of mutation at either Ser 83 or at Asp 87 in all the NAR and NAI strains. None of the NAS strains had a mutation, suggesting that the gyrA gene mutation is sufficient to confer resistance to nalidixic acid and reduced susceptibility to ciprofloxacin. This mutation, although phenotypically expressed as decreased susceptibility to ciprofloxacin, goes undetected by the disk diffusion method using the present NCCLS guidelines. Hence, it can increase morbidity and mortality due to delay in appropriate antibiotic treatment.